Provided are lipid particles encapsulating a nucleic acid capable of expressing an E6 antigen and an E7 antigen of human papillomavirus, whereby a vaccine for preventing and/or treating infection with human papillomavirus type 6 and/or type 11 can be provided. The lipid particles comprise a lipid that is a cationic lipid having the general formula (Ia), or a pharmaceutically acceptable salt thereof. [In the formula, R, R, p, Land Lare as defined in the specification.]
Legal claims defining the scope of protection, as filed with the USPTO.
. The particle according to, wherein each of Rand Rin the general formula (Ia) is a methyl group.
. The particle according to, wherein p in the general formula (Ia) is 3.
. The particle according to any one of, wherein Lin the genera formula (Ia) is a C17-C19 alkenyl group, optionally having one or more acetoxy groups.
. The particle according to any one of, wherein Lin the genera formula (Ia) is a C10-C12 alkyl group, optionally having one or more acetoxy groups, or a C10-C19 alkenyl group optionally having one or more acetoxy groups.
. The particle according to any one of, wherein Lin the genera formula (Ia) is a C10-C12 alkyl group, optionally having one or more acetoxy groups, or a C17-C19 alkenyl group optionally having one or more acetoxy groups.
. The particle according to any one of, wherein Lin the general formula (Ia) is a (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or a (8Z,11Z)-heptadecadienyl group.
. The particle according to any one of, wherein Lin the general formula (Ia) is a decyl group, a cis-7-decenyl group, a dodecyl group, or a (R)-11-acetyloxy-cis-8-heptadecenyl group.
. The particle according to, wherein the lipid further comprises an amphipathic lipid, a sterol and a PEG lipid.
. The particle according to, wherein the lipid further comprises an amphipathic lipid, a sterol and a PEG lipid.
. The particle according to, wherein the amphipathic lipid is at least one selected from the group consisting of distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine.
. The particle according to, wherein the amphipathic lipid is at least one selected from the group consisting of distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine.
. The particle according to, wherein the sterol is cholesterol.
. The particle according to, wherein the sterol is cholesterol.
. The particle according to any one of, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycelol methoxypolyethylene glycol and/or N-[methoxypoly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.
. The particle according to any one of, wherein the PEG lipid is 1,2-dimyristoyl-sn-glycelol methoxypolyethylene glycol and/or N-[methoxypoly(ethylene glycol)2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.
. The particle according to any one of, wherein the lipid composition of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid is amphipathic lipid: 5 to 25%, sterol: 10 to 55%, cationic lipid: 40 to 65% and PEG lipid: 1 to 5% on a molar amount basis.
. The particle according to, wherein the proportion of the amphipathic lipid is 10 to 25%.
. The particle according to any one of, wherein the lipid composition of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid is amphipathic lipid: 5 to 15%, sterol: 35 to 50%, cationic lipid: 40 to 55% and PEG lipid: 1 to 3% on a molar amount basis.
. The particle according to, wherein the proportions of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid are 10 to 15%, 35 to 45%, 40 to 50% and 1 to 2.5%, respectively.
. The particle according to, wherein the proportion of the PEG lipid is 1 to 2%.
. The particle according to any one of, wherein the lipid composition of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid is amphipathic lipid: 10 to 25%, sterol: 10 to 50%, cationic lipid: 40 to 65% and PEG lipid: 1 to 3% on a molar amount basis.
. The particle according to, wherein the proportions of the sterol, the cationic lipid and the PEG lipid are 10 to 45%, 42.5 to 65% and 1 to 2.5%, respectively.
. The particle according to, wherein the proportion of the PEG lipid is 1 to 2%.
. The particle according to any one of, wherein the ratio of the total weight of lipids to the weight of the nucleic acid is from 15 to 30.
. The particle according to, wherein the ratio of the total weight of lipids to the weight of the nucleic acid is from 15 to 25.
. The particle according to, wherein the ratio of the total weight of lipids to the weight of the nucleic acid is from 17.5 to 22.5.
. The particle according to any one of, wherein the human papillomavirus is HPV type 6.
. The particle according to, wherein the human papillomavirus is HPV type 6, and the E6 antigen of HPV type 6 consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 12.
. The particle according to, wherein the human papillomavirus is HPV type 6, and the E7 antigen of HPV type 6 consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 13.
. The particle according to any one of, wherein the human papillomavirus is HPV type 6, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of the human papillomavirus encodes a fusion protein of the E6 antigen and the E7 antigen of HPV type 6, which consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 17.
. The particle according to any one of, wherein the human papillomavirus is HPV type 6, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 6 is an mRNA comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (furin cleavage site), a translated region of E7, a 3′ untranslated region (3′-UTR) and a poly A tail (polyA).
. The particle according to, wherein the sequence of the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 6 consists of a nucleotide sequence having an identity of at least 90% with the sequence of SEQ ID NO: 5.
. The particle according to any one of, wherein the human papillomavirus is HPV type 6, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 6 is an mRNA having a structure comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (furin cleavage site), a translated region of E7 and a 3′ untranslated region (3′-UTR).
. The particle according to, wherein the structure comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (furin cleavage site), a translated region of E7 and a 3′ untranslated region (3′-UTR) consists of a nucleotide sequence having an identity of at least 90% with the sequence of residues 1 to 1018 in SEQ ID NO: 5.
. The particle according to any one of, wherein the human papillomavirus is HPV type 11.
. The particle according to, wherein the human papillomavirus is HPV type 11, and the E6 antigen of HPV type 11 consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 14.
. The particle according to, wherein the human papillomavirus is HPV type 11, and the E7 antigen of HPV type 11 consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 15.
. The particle according to any one of, wherein the human papillomavirus is HPV type 11, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of the human papillomavirus encodes a fusion protein of the E6 antigen and the E7 antigen of HPV type 11, which consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 18.
. The particle according to any one of, wherein the human papillomavirus is HPV type 11, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 11 is an mRNA comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (furin cleavage site), a translated region of E7, a 3′ untranslated region (3′-UTR) and a poly A tail (polyA).
. The particle according to, wherein the sequence of the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 11 consists of a nucleotide sequence having an identity of at least 90% with the sequence of SEQ ID NO: 8.
. The particle according to any one of, wherein the human papillomavirus is HPV type 11, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 11 is an mRNA having a structure comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (furin cleavage site), a translated region of E7 and a 3′ untranslated region (3′-UTR).
. The particle according to, wherein the structure comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6, a protease cleavage sequence (furin cleavage site), a translated region of E7 and a 3′ untranslated region (3′-UTR) consists of a nucleotide sequence having an identity of at least 90% with the sequence of residues 1 to 1018 in SEQ ID NO: 8.
. The particle according to any one of, wherein the human papillomavirus is HPV type 6 and type 11.
. The particle according to, wherein the human papillomavirus is HPV type 6 and type 11, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of the human papillomavirus encodes a fusion protein of the E6 antigen and the E7 antigen of HPV type 6 and the E6 antigen and the E7 antigen of HPV type 11, which consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 19.
. The particle according to, wherein the human papillomavirus is HPV type 6 and type 11, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 6 and the E6 antigen and the E7 antigen of HPV type 11 is an mRNA comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6 of HPV type 6, a translated region of E7 of HPV type 6, a protease cleavage sequence (furin cleavage site), a translated region of E6 of HPV type 11, a translated region of E7 of HPV type 11, a 3′ untranslated region (3′-UTR) and a poly A tail (polyA).
. The particle according to, wherein the sequence of the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 6 and the E6 antigen and the E7 antigen of HPV type 11 consists of a nucleotide sequence having an identity of at least 90% with the sequence of SEQ ID NO: 11.
. The particle according to, wherein the human papillomavirus is HPV type 6 and type 11, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of HPV type 6 and the E6 antigen and the E7 antigen of HPV type 11 is an mRNA having a structure comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6 of HPV type 6, a translated region of E7 of HPV type 6, a protease cleavage sequence (furin cleavage site), a translated region of E6 of HPV type 11, a translated region of E7 of HPV type 11 and a 3′ untranslated region (3′-UTR).
. The particle according to, wherein the structure comprising a cap structure (Cap), a 5′ untranslated region (5′-UTR), a leader sequence, a translated region of E6 of HPV type 6, a translated region of E7 of HPV type 6, a protease cleavage sequence (furin cleavage site), a translated region of E6 of HPV type 11, a translated region of E7 of HPV type 11 and a 3′ untranslated region (3′-UTR) consists of a nucleotide sequence having an identity of at least 90% with the sequence of residues 1 to 1798 in SEQ ID NO: 11.
. The particle according to any one of, wherein the nucleic acid comprises at least one modified nucleotide.
. The particle according to, wherein the modified nucleotide comprises at least one of pyrimidine nucleotide substituted at the 5-position and/or pseudouridine optionally substituted at the 1-position.
. The particle according to, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methoxyuridine, 5-methyluridine, pseudouridine and 1-alkylpseudouridine.
. The particle according to, wherein the modified nucleotide comprises at least one selected from the group consisting of 5-methylcytidine, 5-methyluridine and 1-methylpseudouridine.
. The particle according to any one of, which has an average particle size of 30 to 300 nm.
. Use of the particle according to any one of, for producing a composition for preventing and/or treating infection with human papillomavirus.
. Use of the particle according to any one of, for producing a composition for preventing and/or treating a disease caused by infection with human papillomavirus.
. The use of the particle according to, wherein the disease caused by infection with human papillomavirus is recurrent respiratory papillomatosis or condyloma acuminatum.
. The use of the particle according to any one of, wherein the infection is caused by human papillomavirus of type 6 or type 11.
. A composition comprising the particle according to any one of.
. The composition according tofor expressing the E6 antigen and the E7 antigen of human papillomavirus in vivo or in vitro.
. The composition according tofor use as a medicament.
. The composition according tofor inducing an immune reaction against human papillomavirus.
. The composition according tofor preventing and/or treating infection with human papillomavirus.
. The composition according tofor preventing and/or treating a disease caused by infection with human papillomavirus.
. The composition according to, wherein the disease caused by infection with human papillomavirus is recurrent respiratory papillomatosis or condyloma acuminatum.
. The composition according to any one of, wherein the infection is caused by human papillomavirus of type 6 or type 11.
. A method for expressing an E6 antigen and an E7 antigen of human papillomavirus in vitro, comprising introducing the composition according tointo cells.
. A method for expressing an E6 antigen and an E7 antigen of human papillomavirus in vivo, comprising administering the composition according to any one ofto a mammal.
. A method for inducing an immune reaction against human papillomavirus, comprising administering the composition according toto a mammal.
. A method for preventing and/or treating infection with human papillomavirus, comprising administering the composition according to any one ofto a mammal.
Complete technical specification and implementation details from the patent document.
The present invention relates to a nucleic acid lipid particle vaccine encapsulating mRNA encoding the antigens of human papillomavirus (HPV) type 6 and/or type 11.
Human papillomavirus (HPV) is a virus having circular double-stranded DNA as a genome without envelope membrane, and has about 200 genotypes (Non Patent Literature 1). Some of the genotypes turn infected cells cancerous, and in particular, genotypes 16 and 18, which have been demonstrated to correlate with onset of cancers, including uterine cervical cancer, are classified as high-risk types (Non Patent Literature 2). On the other hand, most other genotypes, such as genotypes 6 and 11, cause epithelial hyperplasia, which is benign and rarely leads to a malignant tumor, and therefore these genotypes are classified as low-risk types (Non Patent Literature 3).
In the genome of HPV, eight viral proteins are encoded, which are classified into early genes (E1, E2, E4, E5, E6 and E7) and late genes (L1 and L2) depending on the time of expression in the lifecycle of the virus. The early genes control virus replication and canceration of infected cells, and L1 and L2 are structural proteins forming outer shells (capsids) of virus particles (Non Patent Literature 4).
Virus proteins E6 and E7 in high-risk type HPV cause abnormal cell proliferation of infected cells. This is because the functions of p53, engaged in induction of apoptotic cell death, and pRb, one of Rb family proteins having a cell cycle regulating function, are inhibited by E6 and E7 (Non Patent Literatures 5 and 6). Even for low-risk type HPV, it has been suggested that p53 is inhibited by E6, and p130, which performs a cell cycle regulating function mainly on the epithelial side, is inhibited by E7 (Non Patent Literature 3). Furthermore, regions important for canceration activity of E6 and E7 have been revealed, and in the case where E6 and E7 are used as vaccine antigens, an inactivating mutation can be inserted to enhance safety (Non Patent Literatures 7 to 9).
The HPV genotypes 6 and 11 infect basal cells through wounded regions in the mucosal epithelia of the respiratory tract, the genital organs and the like, but are normally eliminated by the host immune system. However, in a state of increased susceptibility to infection, proliferation of mucosal epithelial cells is induced by virus proteins E6 and E7, so that a papilloma is formed. This may lead to recurrent respiratory papillomatosis or condyloma acuminatum. On rare occasions, a malignant change may occur (Non Patent Literature 3).
HPV infection is initiated as the L1 protein, forming a capsid, adsorbs to heparan sulfate proteoglycan, present on the surfaces of host cell membranes (Non Patent Literature 10). Neutralizing antibodies, engaged in protection against HIV infection by targeting the L1 protein, are currently marketed as prophylactic vaccines that contain a virus-like particle (VLP) antigen of the L1 protein as a medicinal ingredient. The currently available prophylactic vaccines also contain L1 VLP antigens of genotypes 6 and 11, so that the frequency of surgical operations in treatment of recurrent respiratory papillomatosis is reduced (Non Patent Literature 11).
The host protective immunity against HPV infection consists of induction of a neutralizing antibody and cytotoxic T cells (CTLs) or helper T cells. In particular, nonstructural proteins E6 and E7 are target antigens in CTL induction, and attract attention as therapeutic vaccine antigens for uterine cervical cancer and cervical dysplasia caused by HPV infection (Non Patent Literature 12). Also, these nonstructural proteins attract attention as therapeutic vaccine antigens for the treatment of recurrent respiratory papillomatosis (Non Patent Literature 13).
Patent Literature 1 discloses an E6 and E7 fusion antigen gene sequence of HPV genotypes 6, 11, 16, 18, 31, 33, 39, 45, 52 and 58. In the gene sequence disclosed in the literature, an IgE leader sequence is added to the N-terminus of E6, and a furin peptidase cleavage site is inserted between the translated region sequences of E6 and E7. A mutation is inserted between the p53 binding region of E6 and the pRb binding region of E7 to eliminate the canceration activity of E6 and E7. The gene sequence is introduced into a mammalian expression plasmid to form a DNA gene vaccine against HPV, and its drug effect in a mouse model is evaluated. The immunization is performed by intramuscular administration into the mouse's femoral area by electroporation.
An object of the present invention is to provide a vaccine for preventing and/or treating infection with human papillomavirus type 6 and/or type 11.
The present inventors have found that by administrating lipid particles encapsulating mRNA encoding E6-E7 antigens of human papillomavirus type 6 and/or type 11 to mice, an immune response specific to the antigens is induced, leading to completion of the present invention.
The gist of the present invention is as follows.
(1) A lipid particle encapsulating a nucleic acid capable of expressing an E6 antigen and an E7 antigen of human papillomavirus, wherein a lipid comprises a cationic lipid having the general formula (Ia), or a pharmaceutically acceptable salt thereof:
(2) The particle according to (1), wherein each of Rand Rin the general formula (Ia) is a methyl group.
(3) The particle according to (1) or (2), wherein p in the general formula (Ia) is 3.
(4) The particle according to any one of (1) to (3), wherein Lin the general formula (Ia) is a C17-C19 alkenyl group, optionally having one or more acetoxy groups.
(5) The particle according to any one of (1) to (4), wherein Lin the general formula (Ia) is a C10-C12 alkyl group, optionally having one or more acetoxy groups, or a C10-C19 alkenyl group, optionally having one or more acetoxy groups.
(6) The particle according to any one of (1) to (4), wherein Lin the general formula (Ia) is a C10-C12 alkyl group optionally having one or more acetoxy groups, or a C17-C19 alkenyl group, optionally having one or more acetoxy groups.
(7) The particle according to any one of (1) to (6), wherein Lin the general formula (Ia) is a (R)-11-acetyloxy-cis-8-heptadecenyl group, a cis-8-heptadecenyl group, or a (8Z,11Z)-heptadecadienyl group.
(8) The particle according to any one of (1) to (7), wherein Lin the general formula (Ia) is a decyl group, a cis-7-decenyl group, a dodecyl group, or a (R)-11-acetyloxy-cis-8-heptadecenyl group.
(9) The particle according to (1), wherein the cationic lipid has the following structural formula:
(10) The particle according to (1), wherein the cationic lipid has the following structural formula:
(11) The particle according to (1), wherein the cationic lipid has the following structural formula:
(12) The particle according to (9) or (10), wherein the lipid further comprises an amphipathic lipid, a sterol and a PEG lipid.
(13) The particle according to (11), wherein the lipid further comprises an amphipathic lipid, a sterol and a PEG lipid.
(14) The particle according to (12), wherein the amphipathic lipid is at least one selected from the group consisting of distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine.
(15) The particle according to (13), wherein the amphipathic lipid is at least one selected from the group consisting of distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine.
(16) The particle according to (12) or (14), wherein the sterol is cholesterol.
(17) The particle according to (13) or (15), wherein the sterol is cholesterol.
(18) The particle according to any one of (12), (14) and (16), wherein the PEG lipid is 1,2-dimyristoyl-sn-glycelol methoxypolyethylene glycol and/or N-[methoxypoly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.
(19) The particle according to any one of (13), (15) and (17), wherein the PEG lipid is 1,2-dimyristoyl-sn-glycelol methoxypolyethylene glycol and/or N-[methoxypoly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine.
(20) The particle according to any one of (12) to (19), wherein the lipid composition of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid is amphipathic lipid: 5 to 25%, sterol: 10 to 55%, cationic lipid: 40 to 65% and PEG lipid: 1 to 5% on a molar amount basis.
(21) The particle according to (20), wherein the proportion of the amphipathic lipid is 10 to 25%.
(22) The particle according to any one of (12), (14), (16) and (18), wherein the lipid composition of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid is amphipathic lipid: 5 to 15%, sterol: 35 to 50%, cationic lipid: 40 to 55% and PEG lipid: 1 to 3% on a molar amount basis.
(23) The particle according to (22), wherein the proportions of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid are 10 to 15%, 35 to 45%, 40 to 50% and 1 to 2.5%, respectively.
(24) The particle according to (23), wherein the proportion of the PEG lipid is 1 to 2%.
(25) The particle according to any one of (13), (15), (17) and (19), wherein the lipid composition of the amphipathic lipid, the sterol, the cationic lipid and the PEG lipid is amphipathic lipid: 10 to 25%, sterol: 10 to 50%, cationic lipid: 40 to 65% and PEG lipid: 1 to 3% on a molar amount basis.
(26) The particle according to (25), wherein the proportions of the sterol, the cationic lipid and the PEG lipid are 10 to 45%, 42.5 to 65% and 1 to 2.5%, respectively.
(27) The particle according to (26), wherein the proportion of the PEG lipid is 1 to 2%.
(28) The particle according to any one of (20) to (27), wherein the ratio of the total weight of lipids to the weight of the nucleic acid is from 15 to 30.
(29) The particle according to (28), wherein the ratio of the total weight of lipids to the weight of the nucleic acid is from 15 to 25.
(30) The particle according to (29), wherein the ratio of the total weight of lipids to the weight of the nucleic acid is from 17.5 to 22.5.
(31) The particle according to any one of (1) to (30), wherein the human papillomavirus is HPV type 6.
(32) The particle according to (31), wherein the human papillomavirus is HPV type 6, and the E6 antigen of HPV type 6 consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 12.
(33) The particle according to (31) or (32), wherein the human papillomavirus is HPV type 6, and the E7 antigen of HPV type 6 consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 13.
(34) The particle according to any one of (31) to (33), wherein the human papillomavirus is HPV type 6, and the nucleic acid capable of expressing the E6 antigen and the E7 antigen of the human papillomavirus encodes a fusion protein of the E6 antigen and the E7 antigen of HPV type 6, which consists of an amino acid sequence having an identity of at least 95% with the amino acid sequence of SEQ ID NO: 17.
Unknown
November 20, 2025
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